Rapid Assays for T-Cell Activation by RNA Measurements Using Flow Cytometry

ABSTRACT

The present invention relates to a method for rapidly detecting copies of at least one RNA molecule expressed in individual cells and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No. 61/665,231 filed Jun. 27, 2012 and U.S. Provisional Application No. 61/784,802 filed Mar. 14, 2013. The contents of the applications are incorporated herein by reference in their entirety.

GOVERNMENT INTERESTS

The invention disclosed herein was made at least in part with Government support under Grant Nos. AI045761, AI106036, MH079197, and HL106788 from the National Institutes of Health. The United States government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to methods that rapidly detect gene expression in individual cells.

BACKGROUND

Many pathological conditions provoke T cell-mediated immune responses. These include cancer, autoimmunity, transplantation, and infectious diseases, including, for example, tuberculosis (TB). In all these conditions, activation of T cells involving interaction of a T cell receptor (TCR) with a specific antigen (Ag) as a common denominator, while the functional consequences of this interaction vary. In any given T cell, the TCR signaling cascade may stimulate expression of different sets of genes depending on the physical nature of the Ag, its presentation, the TCR repertoire, and the strength of Ag-induced activation. These induced differences in gene expression patterns during cell activation, proliferation and differentiation may ultimately contribute to functional heterogeneity among the clonally expanded effector cells. An additional element of cell-to-cell diversity arises during T-cell homeostasis. When the Ag is cleared, most expanded effector cells die by apoptosis, while a small fraction of cells survive as long-lived memory cells. The responses that occur upon re-exposure to Ag are governed by the factors listed above, and are further influenced by the prior differentiation process. As a result, for each Ag encountered, the immune response generates functionally distinct subsets of T cells (for example, effector vs. central memory vs. effector memory cells) sharing the same Ag specificity. At any given time in the natural history of an immunopathological condition, the relative representation of these different subsets varies; they may determine or reflect the disease process. T-cell subsets correlate with Ag and pathogen load in vivo and, as exemplified by studies of chronic viral infections and TB, quantitative analysis of these key subsets is promising for diagnostics and measurements of protective immunity. Tell-tale changes in T cell subsets should also be seen during treatment, since treatment outcome is associated with a particular evolution of the immunopathological condition. Such signatures of evolving states (natural or treatment-mediated) escape ensemble measurements and can only be detected at the single T cell level. The properties of individual T cells are only fully revealed by their response to Ag stimulation.

Current, blood-based clinical immunoassays can typically recognize the presence of non-infectious and infectious pathology, but may not reveal progression of the pathology. In contrast, single-cell, functional analysis of Ag-specific immune cells in peripheral blood should unravel multiple, intersecting immunopathological states associated with evolution of infection and disease. Such analysis would help advance prognostic capability and foster effective intervention. A compelling example is provided by infection with Mycobacterium tuberculosis. With current methods, TB is typically diagnosed after the diseased subject has already transmitted infection to his/her contacts. Yet diagnosis of asymptomatic infection per se does not warrant medical intervention. The ability to distinguish stable latent infection (LTBI) from early active disease (before it is contagious) is greatly needed. Existing immunodiagnostic assays fail to address this need, as they are not geared to distinguish between Ag-specific, functional T cell subsets known to differ between infection states. Moreover, these assays do not assess responses that may reflect stage-specific expression of mycobacterial Ags.

The hallmark of M. tuberculosis infection is the huge number of people infected asymptomatically with this pathogen (2 billion). In the absence of immunocompromise, 90-95% of latently infected individuals will not develop active TB. However, given the size of the reservoir, M. tuberculosis infection still causes 9.4 million new cases of active TB and 1.7 million deaths per year. Transmission of infection would be greatly reduced if it were possible to identify and treat infected individuals as they progress to active disease before they become symptomatic and infectious. Detecting tubercle bacilli or bacillary products is exceedingly difficult during early active disease, due to very low bacillary numbers. The standard, microbiological diagnosis of active TB detects individuals who already bear tubercle bacilli in their respiratory secretion and are therefore infectious.

Methods based on ensemble measurements are simply not geared to revealing the complexity of the cell-mediated immune response and cannot reveal rare or transient cell states associated with disease stage and evolution. The most common immunoassay formats for single T cell analysis are the enzyme-linked immunospot assay (ELISPOT) method and quantitative flow cytometric (FC) analysis. In a commercial ELISPOT test from Oxford Immunotech (United Kingdom), peripheral blood mononuclear cells (PBMCs) are first isolated from a blood sample, washed and counted. Then a predetermined number (such as 250,000) of PBMCs and M. tuberculosis-specific Ags are added to plate wells pre-coated with antibodies to interferon gamma (IFNγ) and incubated overnight (16-20 hours). IFNγ released from activated T cells is captured in the wells. This is followed by incubation with a second antibody (Ab) conjugated to a color-forming enzyme, after which the wells are washed and a color-forming substrate is added. Spots are produced where IFNγ was secreted by T cells giving a characteristic appearance of a colored ring surrounding the cytokine-releasing cell. Finally, spots are counted, either by naked eye or using a plate reader. ELISPOT is highly sensitive (its detection limit is 10/10⁶ in PBMCs). However, it fails to provide the desired discrimination among functional T cell subsets for several reasons. First, it is not typically amenable to simultaneous detection of several targets. Second, the format limits the number of cells for analysis (e.g., 250,000 cells per well in the commercial T-SPOT.TB test mentioned above). Conventional FC has multi-parameter capability. Cells in whole blood or PBMCs are stimulated by incubation for 6 hours or more with specific Ags, for example M tuberculosis-specific peptides or proteins, and extracellular secretion inhibitor such as brefeldin or monensin. Permeabilized T cells are then stained with fluorophor-labeled anti-cytokine Ab (for example, FITC-conjugated anti-IFNγ) and analyzed by FC. Staining of proteins with Ab reduces the versatility of conventional FC, particularly in the case of intracellular proteins. Moreover, assays are limited by protocols that require prolonged stimulation of T cells by endogenous antigen presenting cells (APCs), which increases turn-around time and reduces clinical usefulness.

The existing methods for cytokine analysis of individual cells lack the ability to rapidly identify low-frequency T cell populations present in small samples of peripheral blood. The commercial ELISPOT test does not distinguish between TB and LTBI. Moreover, a positive result in an asymptomatic individual (in the absence of microbiological evidence of active TB) is uninformative for reactivation risk. Thus, while accurately reflecting the presence of M. tuberculosis infection, a positive result is not considered an indication for therapeutic intervention. This diminishes the acceptability of the test, particularly in low-resource, high-burden countries, where most of the population is infected (up to 80% in regions of South Africa). Moreover, even in low burden, high-resource countries such as the US, the side effects of LTBI treatment often lead to its refusal when it is recommended without an indication of reactivation risk. Clearly, the limitations of current immunodiagnostics have enormous public health consequences worldwide.

Thus, there is an unmet need for methods that rapidly detect gene expression in individual cells.

SUMMARY

This invention relates to novel rapid methods for detecting various RNA molecules in individual cells.

Accordingly, one aspect of this invention provides a method for detecting copies of at least one RNA molecule expressed in individual cells. The method includes (a) providing a sample containing a population of cells (e.g., at least 100 or at least 10,000 cells) that express specific receptors capable of initiating signal cascades leading to gene expression upon binding to them of ligands or stimulating molecules; (b) inducing gene expression in the cells ex vivo, either immediately or after culturing, by incubating the cells with at least one compound, for example, a peptide or mixture of peptides derived from a microorganism (such as M. tuberculosis) that will bind specific receptors or otherwise initiate signaling and initiate gene expression; (c) fixing and permeabilizing the cells; (d) labeling copies of at least one RNA molecule expressed by the cells with a set of fluorescently labeled oligonucleotide hybridization probes, and washing away unbound probes; and (e) detecting cells having expression events by flow cytometry (FC), an expression event being one or more fluorescence measurements categorized by fluorescence intensity gating.

The aforementioned method can be used for detecting various RNA molecules in individual cells. In particular, it can be used for detecting RNA molecules encoded by cytokine genes, such as IL-2, TNFα, or IFNγ.

In the method, the step of inducing can include stimulating T-cell receptor (TCR) signaling and down-stream gene expression by antigen presenting cells (APCs) that are present in the cell population or by artificial APC (aAPC). The induction can be carried out for a period of from 30 minutes to 6 hours. In either case, the step of inducing can also include co-stimulating with at least one monoclonal antibody (mAb). The monoclonal antibody can be bound to or added to the aAPC or APCs.

In one embodiment of the method, the cell population can be a population of T cells obtained from human PBMCs. In that case, the at least one RNA molecule can be one encoded by a cytokine gene. And, cells in the cell population can be induced by APCs or aAPCs with one or more peptide-loaded MHC molecules that interact with TCR.

In the method, the compound or stimulatory molecules can be derived from a microorganism, such as M. tuberculosis-derived immunogenic peptides. In one example, the compound can contain a peptide or mixture of peptides, such as those that associate with MHC class I or MHC class II molecules. These peptide or mixture of peptides can be added to the APC or aAPC mentioned above at various concentrations, e.g., between 1-20 μg/ml.

In the above-described method, the step of inducing can include stimulating toll-like receptor signaling and down-stream gene expression in a cell containing such receptors with a stimulus, examples of which include cytokines, microbial products or synthetic compounds. Examples of the microbial product include a lipid, glycan, glycolipid, sulfolipid, glycoprotein, protein, peptide, or nucleic acid (e.g., RNA or DNA). The stimulation can be carried out for a period of from 30 minutes to 72 hours (e.g., 30 minutes to 6 hours, 6 hours to 24 hours, 24 hours to 72 hours). The stimulus can be present at a concentration between 1-20 μg/ml (e.g., 1-100 ng/ml or 100-1000 ng/ml). The compound or stimulus can be a cytokine, such as IFNγ, IL-2, IL-15, TNFα, or a cytokine other than IFNγ, IL-2, IL-15, or TNFα. In the method, more than one compound or stimulus can be provided either at the same time or at different times.

In step (d) of the method, the probe set can include 20-60 probes, each singly labeled with the same fluorescent moiety.

In another embodiment, the detected cells having expression events can be separated (e.g., by fluorescence-activated cell sorting) from cells not having expression events. The separated cells can include only one cell or more than one cell (e.g., 10, 100, 1000, 10,000 or 100,000 cells). Gene expression can then be measured in the separated cells by, e.g., RT-PCR or a transcriptomic analysis of RNA in the cells.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of fluorescence intensity versus forward scattered channel A (FSC-A) for the FC readouts described in Example 2.

FIG. 2 is a set of four frequency plots from FC detection of samples described in Example 7.

FIG. 3 is a set of six frequency plots from FC detection of samples described in Example 8.

FIG. 4 is a set of three frequency plots from FC detection of samples described in Example 9.

FIG. 5 is a set of twelve frequency plots from FC detection of samples described in Example 10.

FIGS. 6A and 6B are (A) a set of twenty-eight frequency plots from FC detection of samples probed with mRNA probe sets as described in Example 11 and (B) a set of sixteen frequency plots from FC detection of samples probed with mAbs as described in Example 11.

FIGS. 7A and 7B are (A) a set of two frequency plots from FC detection of samples probed with a probe for green fluorescent protein, before (left panel) and after (right panel) additional gating to distinguish the cells as cytokine-positive and cytokine-negative populations, as described in Example 12 and (B) a set of two frequency plots from FC detection of samples probed simultaneously with four mRNA probe sets, before (left panel) and after (right panel) additional gating to distinguish the cells as cytokine-positive and cytokine-negative populations, as described in Example 12.

FIGS. 8A and 8B are (A) a table showing the threshold cycles of amplifications of two genes in the sorted (+/−) cell populations described in Example 12 and (B) a photograph showing an electrophoretic gel of the amplification products from the amplifications described in Example 12.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses a method for measuring and assessing expression of one or more genes, including RNA transcripts that code for proteins, for example, messenger RNA (mRNA) and pre-mRNA, and those that do not.

As disclosed herein, the method can be used to assessing RNA transcripts in cells the gene expression of which changes in response to various inductions. Induction may be naturally occurring or stimulated by any known ligands for cellular receptors including but not limited to TCRs and involve, in principle, any receptor or ligand (for example, Toll receptor-like or chemokine receptors and their ligands) or compound that initiates a signaling cascade and may result in transcript synthesis and expression of certain genes, for example, at least one cytokine gene such as IL-2, TNFα, or IFNγ.

In some embodiments, the induction is stimulation by a compound or compounds that bind specific receptors and thereby initiate gene expression, for example, a peptide or mixture of peptides associated with MHC I or MHC II molecules. The compound or compounds may be derived from a microorganism, for example, stimulatory molecules that are M. tuberculosis-derived immunogenic peptides. Induction of gene expression, for example stimulation of T-cell receptor signaling and downstream gene expression may be by APCs that are present in the cell population being investigated. Alternatively, stimulation may be by artificial APC (aAPC). A peptide or mixture of peptides that associate with MHC class I or class II molecules may be added to APC or aAPC, preferably at a concentration in the range of 1-20 μg/ml to produce peptide-loaded MHC molecules that interact with TCRs. Certain embodiments of methods of this invention include co-stimulation with at least one monoclonal antibody in addition to APC or aAPC. With aAPC, the mAb may be bound to the aAPCs.

The method of this invention allows one to carry out rapid and sensitive measurements of changes in cell population composition and function that occur during evolution of infectious or non-infectious disease and associated progression of pathology. These measurements can characterize the properties of neoplastic and/or malignant cells of hematopoietic and non-hematopoietic origin, and of cells involved in autoimmune disease.

The method can also be used in monitoring of response during therapy to treat the disease state. For infectious disease, immune control in response to antibiotic therapy can be determined by correlating progress in eradicating a pathogen, for example M tuberculosis, with changes in cellular responses to stimulation, such as responses of functional T-cell subsets to antigenic peptides.

Methods of this invention are generally applicable to virtually all cells, mammalian (including but not limited to human) and lower eukaryotic species, that express specific receptors capable of intiating signaling cascades upon ligand/stimulatory molecule binding. They can be applied to populations of bacteria as well as to multicellular organisms. Certain preferred embodiments are the application of methods of this invention to animal cells, including but not limited to human cells. A suitable source provides a sufficient number of cells for detection of rare events and/or for statistical analysis. In some embodiments that is as few as 1000 or even 100 cells. In many embodiments, for example where the cells are T cells, a source that provides at least 10,000 cells is preferred. More preferably, the source provides at least 100,000 cells or at least one million cells. Suitable sources include blood samples and tissue samples, for example, biopsy samples. Tissue samples must be separated into individual cells, that is, disaggregated tissue is required for processing.

As mentioned above, methods of this invention include rapid induction, not exceeding 5 days and preferably less, for example 4, 3, 2, or 1 day, or 16, 12, 10, 8, 6, or 4 hours. A preferred induction time is from 30 minutes to 8 hours, preferably from 30 minutes to 6 hours, more preferably from 30 minutes to 4 hours, and even more preferably from 30 minutes to 2 hours.

Methods of this invention include rapidly measuring expression of one or more genes in immune and non-immune cells based on activation and functional consequences induced by ion fluxes, including but not limited to those caused by ionophores and/or mitogens, or by signaling through cell surface or intracellular receptors, including but not limited to pattern recognition receptors, including toll-like receptors and NOD and NOD-Like receptors, G protein coupled receptors, including chemokine receptors, polypeptide hormone receptors, cytokine receptors, B cell receptors, or TCR. Methods according to this invention are believed to advance medical care for various infectious and non-infectious pathologies. Preferred embodiments comprise detecting expression of RNA, including both mRNA and pre-mRNA, for one or more cytokines in individual lymphocytes, whether in isolated PBMC, T cells, or in whole-blood samples (rather than isolated PBMC) and also other types of cells as indicated below. Methods according to this invention apply, for example, to analyzing CD3⁺CD4⁺ T cells (T helper 1 or Th1 cells) that produce IL-2, IFNγ, and TNFα; CD3⁺CD4⁺ T cells (T helper 2 or Th2 cells) that produce IL-4, IL-5, IL-6, IL-10, and IL-13; CD3+CD8+ T cells that produce (IL-2, IFNγ, TNFα, MIP-1α and other chemokines) and other specialized T-cell subsets, including but not limited to Th17 and Treg cells, or other lymphocyte subsets, including but not limited to NK cells, NKT cells, subpopulations of macrophages and dendritic cells, cells comprising endothelia and epithelia of various body organs or cells of the nervous system, including but not limited to neurons and glia, and the pre-mRNA and mRNA for cytokines, chemokines and functional molecules (for example, see Table 2) produced by these cells.

Certain methods according to this invention comprise ex vivo stimulation of cells, or induction of specific molecular interactions leading to expression of activation markers and modulating cellular functions. Stimulation may be performed immediately or performed on cells after culturing in a culture medium. Stimulation may be natural, that is, simply incubating the cells in culture. Preferably, stimulation may be promoted by incubating the cells in culture with one or more compounds that trigger specific receptors/ligands and induce signaling cascades. These compounds may be synthetic or derived from microorganisms, including but not limited to bacteria, viruses and fungi, for example, lipopolysaccharides, lipoarabinomannans, peptidoglycans, mycolic acids of bacterial origin, or proteins of viral origin, including but not limited to Epstein Barr virus or cytomegalovirus (for example, proteins ebvIL-10, cmvIL-10 and UL146, respectively), or fungal (for example, Cryptococcus- and Aspergillus-associated) galactoxylo- and galactomannans and soluble antigens; polypeptide hormones, for example, platelet-derived growth factor or VEGF; cytokines; and antigens, including antigenic peptides. A preferred method of stimulation is with artificial antigen presenting cells (aAPC; see below).

Methods according to this invention further include detection of individual cells, including particularly activated cells, having a biomarker signature, preferably a multiple biomarker signature, namely one or more RNAs, including particularly mRNAs or pre-mRNAs in situ in intact cells.

Preferred methods according to this invention comprise detecting induced gene expression, for example for cytokines, and most preferably for multiple cytokines, in individual cells, for example activated T cells, by labeling expressed RNA molecules, including mRNA or pre-mRNA molecules, in fixed, permeabilized cells utilizing sets of fluorescently labeled hybridization probes and washing away unbound probes. Single-molecule sensitivity is obtained by employing multiple nucleic acid hybridization probes that provide multiple fluorescent labels for each RNA target. These may be a small number of probes multiply labeled with a particular fluorophore or probes labeled to produce fluorescence by fluorescence resonance energy transfer (FRET) when they are hybridized to cognate mRNA or pre-mRNA. Preferred methods of this invention utilize a larger number, for example, 10-100, more preferably 20-60, even more preferably 30-50, for example about 50, shorter oligonucleotide probes, each labeled with a single fluorescent dye, that bind simultaneously to a target sequence. The attachment of many labels to each RNA molecule renders the cell sufficiently fluorescent above background that it can be detected by FC methods.

Further, methods according to this invention include detection and analysis of individual cells expressing target RNA or RNAs by FC. Certain preferred methods of this invention comprise using quantitative FC to detect cells that include RNA expression products of cytokine genes induced in desired cells by APC or aAPC stimulation.

FC comprises a fluidics system for hydrodynamic focusing to create a single file of cells. Single cells can then be interrogated for fluorescence emission at one or multiple wavelengths. FC is a routine technique in clinical pathology laboratories. For example, immune phenotyping by FC is an important tool in the diagnosis and staging of various haematologic neoplasms. Essential FC steps are labeling cells with one or more fluorescent moieties, for example fluorophores, introducing labeled cells into a flow cytometer, illuminating each cell with an excitation light source such as a laser that emits at a wavelength that excites the fluorescent moieties, and detecting emitted fluorescent light using filters and mirrors that distinguish specific emitted wavelengths for each fluorescent moiety being used, so as to acquire data that reveal the presence and, preferably, the amount of each fluorescent moiety associated with each cell based on the excitation and emission.

As disclosed herein, FC can be used for detecting cells expressing RNA by utilization of multiple singly-labeled fluorescent hybridization probes. A most preferred embodiment is detection of RNA (mRNA or pre-mRNA) expressed from cytokine genes in the T-cell fraction of isolated. PBMC that had been stimulated by presentation of M tuberculosis Ags by either endogenous APC or, more preferably, aAPC. RNA for multiple cytokines can be detected by use of probes that are specific for each species of RNA and that are labeled with a different fluorescent moiety (for example, a different fluorophore) for each species of RNA detected.

In methods of this invention, FC is performed on populations of cells (from as few as 10,000 cells to more than one million cells). Data are acquired for one or more events. An event is a set of measurements for one cell. The data for each event are categorized into user-defined windows, for example, signal for fluorophore A, or signal for fluorophore A that is between an intensity level X and an intensity level Y. The categorization of FC readings is a process called gating, and the events are thus gated, or selected, typically as above a certain threshold or bounded by certain threshold limits. Results can then be presented as absolute numbers of cells in a certain window or, preferably, as a fraction of cells in one or more windows, that is, frequency. For example, in analyzing a disease state, the results might be that 10% of the cells were positive for mRNA A and mRNA B, 7% of the cells were positive for mRNA B and mRNA C, and 2% of the cells were positive for all three of mRNA A, mRNA B and mRNA C. If 10,000 cells are analyzed, the lowest possible positive result is that a certain event occurred once in 10,000 cells. If, on the other hand, one million cells are analyzed, the lowest possible positive result is that a certain event occurred once in one million cells.

Analysis of FC results may include analysis of variance (ANOVA), which is a collection of statistical models for analyzing variances, generally to test significant differences between means for groups or variables by partitioning total variances into components due to true random error and components due to differences between means.

This invention also discloses reagent kits for carrying out the foregoing methods. Kits according to this invention include at least one stimulatory compound, for example proteins or peptides that will stimulate expression of one or more cytokines, as a response characteristic of the disease state to be tested, or aAPC that are loaded with such peptides and that carry co-stimulatory molecules as described below, and one or more sets of oligonucleotide probes that each hybridize specifically to a single sequence present in mRNA or pre-mRNA, preferably wherein each set comprises 20-60 oligonucleotides of 16-20 bases each and each oligonucleotide is labeled with a single flourophore. The kit may additionally comprise other than mRNA-specific probes such as fluorophore-conjugated Abs recognizing cellular proteins or chemical probes tagged with fluorophores binding to targets of interest, reagents for fixation and permeabilization of cells to be analyzed, reagents for hybridization of the included probes with the cells, and reagents for post-hybridization processing, such as washes to remove excess, non-hybridized probe(s) or other unbound labeled probes.

In a preferred embodiment, the method of this invention includes stimulation of TCR signaling and down-stream gene expression by APCs that are present in a cell population or by synthetic beads, which are referred to herein as aAPC. Synthetic beads serve as a mechanical support, i.e., a platform to which proteins (MHC and/or co-stimulatory receptors and ligands) can be attached via chemical, charge or any other type of bonding. Therefore, for example, any plastic surface could be used with or without chemical modification or derivatization as necessary, to serve as a mechanical support, or platform, for aAPC.

The APC interact (or react) with added peptides (or proteins) so that they are associated with (or endogenously processed and presented by) MHC molecules and then can stimulate TCR. The aAPC interact with added peptides that bind to MHC molecules and these peptide-loaded aAPC can then stimulate TCR. The aAPC thus carry one or more classes of peptide-loaded MHC molecules to interact with TCR, with or without one or more antibodies or other type of ligands that interact with co-stimulatory receptors expressed on T cells, such as CD28 and/or CD49d. In certain preferred embodiments stimulatory peptides are M. tuberculosis-derived immunogens. The step of stimulating TCRs comprises incubation of TCR-expressing cells with APC or aAPC that comprise MHC class I or class II molecules loaded with appropriate peptides, and, in some embodiments, with co-stimulatory proteins or functional equivalents such as antibodies, peptides, various carbohydrate- and lipid-bearing molecules that bind their targets on co-stimulatory receptors that enhance signaling, resulting in T cell activation.

Functionally different T-cell subsets are often expressed at frequencies close to current detection limits (generally 0.01-0.1%). Thus, their detection requires potent TCR stimulation and highly sensitive detection methods so that even rare T-cell subsets can be directly detected or expanded to numbers above the detection limit of the particular assay method. Methods of this invention comprise inducing effective TCR stimulation by APC that are present in a cell population or by or aAPC, a synthetic bead-based platform containing (i) MHC class I or class II molecules loaded with appropriate peptides and, in certain preferred embodiments, (ii) co-stimulatory proteins (anti-CD28 and/or anti-CD49d monoclonal antibody) that provide additional signals (Oelke et al., 2003, Nature Medicine 9(5):619-24]. Virtually any MHC allele can be attached to the bead platform and any co-stimulatory signal can be provided as a bead-attached specific monoclonal antibody (mAb) or co-receptor ligand, whether naturally occurring or chemically defined. In addition to their superior properties of stimulation, aAPC are very stable: the final cell-sized aAPC, with or without bound synthetic peptides, have a long shelf life in a lyophilized form, and are readily transportable.

In methods of this invention, the procedure for stimulation generally includes obtaining the desired cells from a subject, incubating the cells in culture media, and adding compounds for stimulation of the cultured cells for various times, such as 30 minutes, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, or longer periods (e.g., 1, 2, 3, 4, or 5 days). Cells might be obtained by biopsy of solid tissue or aspiration of cells in liquid from a site of interest, such as by bronchoalveolar lavage, or the source of the cells may be peripheral blood. Desired cells may be cultured after separating them from unwanted cells by lysing those cells and separating the intact desired cells from the remnants of lysed cells, or by separating the desired cells from the undesired cells based on specific cell characteristics. For example, PBMCs from blood may be cultured either after lysis of red blood cells (RBC) or after recovery based on cell density by sedimentation on Ficoll and removal of Ficoll prior to incubating in culture media. Stimulation can be achieved by adding a stimulating compound as described above (such as a bacterial, fungal, or viral product, or a host protein or peptide) in an amount sufficient to produce the response that will be detected and incubating for a time sufficient to produce the response. The response may be synthesis of a pre-mRNA or mRNA by the cultured, stimulated cells. The response may be interpreted as a signature characteristic of the desired cells' response to the stimulus given.

For obtaining signatures of M. tuberculosis infection, for example, HLA-A*0201-based aAPC can be loaded with HLA-A*0201 epitopes (peptides) known to bind to the human HLA-A-0201 MHC class I protein derived from known Ags such as Rv1886c, Rv3874 and Rv3875, and the Ag loaded, A2-based aAPC can be mixed with PBMC at a 1:1 ratio under standard culture conditions for detection of CD8+ T effectors. HLA-DRB1*04-based aAPC can be similarly loaded and used for detection of CD4+ T effectors.

For detection of cells containing copies of one or more specific RNAs, one can utilize for each RNA a set of multiple, fluorescently labeled, oligonucleotide hybridization probes. The oligonucleotide probes may be DNA, RNA, or mixtures of DNA and RNA. They may include non-natural nucleotides, nucleotide analogs and non-natural inter-nucleotide linkages. Each probe may be labeled with multiple fluorescent moieties, for example, multiple copies of a fluorophore, Quantum Dot, or other fluorescent moiety. For example, WO/1997/014816 describes detection of beta- and gamma-actin mRNAs by a single-step in situ hybridization utilizing five probes per target, the probes being about 50-nucleotide long single-stranded DNA labeled with a fluorophore (Fluorescein or Cy3) every tenth nucleotide, that is, five fluorophores per probe. Preferred methods of this invention utilize a larger number, for example, 10-100, more preferably 20-60, even more preferably 30-50, for example about 50, shorter oligonucleotide probes, each labeled with a single fluorescent dye, that bind simultaneously to a target sequence. The attachment of many labels to each RNA molecule renders the cell sufficiently fluorescent above background that it can be detected by FC methods. In most embodiments, each probe set will have a single fluorescent moiety, and each fluorescent moiety will be detectably distinguishable from other fluorescent moieties that are present. If one is detecting whether or not each cell expresses a first RNA or a second RNA, however, both probe sets could be labeled with the same fluorescent moiety.

Background fluorescence presents a different problem for methods of this invention, which utilize FC, than from microscopic smFISH methods, which utilize microscopic visualization of individual RNAs as bright spots in cells. Methods of this invention may employ background reduction techniques. One such technique is to employ FRET between probes that align adjacently on a target RNA. Considering, for example, a first probe and a second probe that align adjacently with the 5′ end of the first probe adjacent (within appropriate FRET distance, as is known) to the 3′ end of the second probe, one may add a FRET donor to the 5′ end of the first probe and add a FRET acceptor to the 3′ end of the second probe. With this combination, cells are excited at the absorption wavelength of the donor fluorophore, for example Fluorescein, but signal is detected at the emission wavelength of the acceptor fluorophore, for example Texas Red. Because Texas-Red fluorophores are not excited directly, unhybridized probes or mis-hybridized probes do not fluoresce. Another technique is to employ FRET between a double-strand DNA dye and a fluorophore of a probe set, as is performed in probing with Resonsense probes. In this case a dsDNA dye such as SYBR Green is included (SYBR Green has absorption and emission wavelengths very similar to Fluorescein), and a probe set is labeled with a fluorophore that accepts emission from SYBR Green, for example, TMR. With this combination one can excite the cells at the absorption wavelength of SYBR Green, but detect emission at the emission wavelength of the fluorophore. Because the fluorophore is not excited directly, unhybridized probes do not fluoresce.

Although all regions of an RNA can serve as targets for probes, several factors should be considered in the selection of such probes. Preferably the target region should not be expressed in the cell from other regions of the genome, or more preferably, the target region should not be present elsewhere in the genome. This can be ensured by checking the sequence of the desired target against the databases that list the expressed genes and the entire genome sequences for the organism being studied. This “filtration” of the potentially background-generating sequences can be performed using publically available computer programs such as “repeat masker.” The probes can be selected from immediately adjacent regions of the targets or there can be some space between adjacent probes. The length of the probes can vary depending upon the stringency of hybridization.

As shown below, Example 1 demonstrates induction and probing in distinguishing characteristic signatures of gene expression in single cells.

More specifically, assays were carried out to test differentiated THP-1 cells stimulated with irradiated M. tuberculosis. Cells were fixed by incubation for 20 minutes with 4% paraformaldehyde after 24 hours of stimulation and then hybridized with probes for two mRNA targets. One was TNFα, a key target for analysis of activated T cells and macrophages. The other was ACSL1 (a lipid metabolism gene, GenBank Accession # BC050073.1), which is also expected to be induced by the stimulus. The probe set specific for TNFα (Ensembl Sequence ID ENST00000376122) comprised 48 probes, each about 20 nucleotides long and terminally labeled with a tetramethylrhodamine fluorophore. The probe set specific for ACSL1 comprised 48 probes, each about 20 nucleotides long and labeled with an optically distinguishable fluorophore, namely, AlexaFluor 596.

For Example 1, expressed RNA products were detected using the known microscopic technique described in Raj et al., 2010, Methods in Enzymology 472:365-386; and Raj et al., 2008 Nature Methods 5: 877-879, namely, smFISH. This technique comprises detection of fluorescent spots as an indication of hybridization of a probe set to an individual RNA molecule. In Example 1, spots corresponding to both mRNAs were detected. Images for TNFα and ACSL1 were merged 3-D stacks for each channel for the same set of cells. Interpretation comprised image processing using a computer program to identify the diffraction-limited spots, corresponding to the individual molecules of mRNA, overlaying them on a DIC image of the cells, and obtaining a cell-by-cell count of the number of mRNA molecules, as described in the references. A large cell-to-cell variation in the number of transcripts for each gene was observed, consistent with previous observation that mRNA synthesis in mammalian cells is highly stochastic. The results, the average spot number for TNFα in stimulated and unstimulated cells based on the total number of spots counted in 50 consecutively analyzed cells, showed that single cell measurements are far more informative than ensemble measurements.

FC data acquisition for methods of this invention can be carried out by conventional FC techniques. Light can be detected in both a forward scatter channel (FSC) and a side scatter channel (SSC). Data can be presented as either or both of density plots and contour diagrams. To visualize cells of interest while eliminating results from unwanted particles, for example debris, FC gating is used. Gates and windows, or regions, are determined in the conventional manner such that positive events appear in a window. To assist in gating to discriminate true positive events from false positives, use of a negative control, that is, probing for something that the cells could not express, is helpful. In principle this is analogous to FC for proteins, where an isotype negative control and/or unstained cells are utilized.

FC detection reveals the number of positive events in a sample, for example, one positive event in 10,000 cells (1/10,000), three positive events in 100,000 cells (3/100,000), or two positive events in one million cells (2/10⁶). The frequency of an event may lead to a conclusion as to a biological state, for example, a disease state. Alternatively, combinations may do so; for example, 10% of cells in a sample are positive for a first cytokine (cytokine A) and also positive for a second cytokine (cytokine B). As another example, a disease state might be characterized by the following combination: 10% or more of cells being positive for cytokines A and B; 7% or more of cells being positive for cytokine B and a third cytokine (cytokine C); and 2% or more of cells being positive for all three cytokines.

As disclosed herein, it was demonstrated that FC is useful as a read-out for methods of this invention. The experiment described below in Example 2 was designed to test whether signals generated by probe sets hybridized to RNA targets in individual cells can be detected by FC. That example describes steps to detect HIV GAG mRNA in cell cultures expressing it from a lentiviral construct. More specifically, 293T cells were transfected with three plasmids that together allow expression of a recombinant lentiviral construct and hybridized with probes specific for GAG mRNA. FC results are presented in FIG. 1, graphs of fluorescence intensity versus forward scattered channel A (FSC-A) obtained from cells that were not transfected, on the left, and cells transfected with the three plasmids from a lentiviral packaging system, on the right. Both sets of cells were hybridized with a probe set for GAG mRNA. The population of transfected cells produced signals of intensity that was 1-2 orders of magnitude greater than seen with the signals from the outlier untransfected cells (i.e., those above the set threshold). As a control, image-based analysis of smFISH results indicated that about 25% of the cells were expressing the construct. The FC analysis on the same population found the same fraction of cells to be fluorescent. These results demonstrate that mRNA expression is readily detected by FC. Moreover, the data provide guidance for distinguishing a low frequency of strongly stimulated (brightly fluorescent) T cells from outliers in the non-stimulated population. Receiver operating curve (ROC) analysis can define an optimal threshold. ROC analysis is well known to those versed in the art of test development. See, for example, Zweig et al., 1993, Clinical Chemistry 39 (8): 561-577 and Pepe, 2003, The statistical evaluation of medical tests for classification and prediction. New York, N.Y.: Oxford.

The challenges in the detection of transcriptional signatures of T cell activation are two fold—only a small subset of cells are induced and the induced cells express only a few copies of mRNAs per cell. In experiments described in Example 7, the inventors were able to demonstrate both sensitive detection of low frequency responses and simultaneous detection of several mRNA targets by methods of this invention. The assay, which included stimulation, probing of fixed cells and FC, was able to detect cells expressing the IL-2/IFNγ pair with frequency of 3.46% (FIG. 2, right panel), while cells expressing IL-2/TNFα and IFNγ/TNFα cytokine pairs were detected with frequencies of 3.35% and 6.37%, respectively. Comparisons of single- and double-cytokine populations, e.g., IFNγ (4.76-5.40%) vs. IL-2/IFNγ (3.46%), suggested that not all IFNγ producers could produce IL-2. In contrast, most IFNγ producers (5.40%) seemed to also express TNFα, as suggested by the size of IFNγ/TNFα-producing population (6.37%). This result is expected for a population of T cells non-specifically activated by combined TCR triggering and lectin-mediated stimulation (anti-CD3 mAb and phytohemagglutinin). Thus, the assay according to this invention gave results consistent with known T cell biology.

As shown in Example 8, a method according to this invention was applied for the detection of M. tuberuclosis-specific cells stimulated ex vivo. PBMC were incubated for 5 h with a mixture of ESAT6- and CFP10-derived peptides (T-SPOT.TB, Oxford Immunotec, UK) at 5 μg/ml. As shown in FIG. 3, there was a ˜2-fold increase in cells expressing cytokine mRNAs in Ag-specific T cells after this stimulation. Therefore, even in the absence of co-stimulation provided by anti-CD28 mAb, the assay detected low frequencies of circulating M. tuberculosis-specific T cells present in this LTBI donor. This result demonstrates the utility of assays according to this invention.

Example 9 describes a method according to this invention with increased signal as compared to Example 8. Engagement of CD28 co-receptor of TCR by mAb was used to deliver superior stimulation of Ag-specific T cells. As shown in Example 8, PBMC were incubated for 5 h with a mixture of ESAT6- and CFP10-derived peptides (T-SPOT.TB, Oxford Immunotec, UK) at 5 μg/ml, but this time with or without anti-CD28 mAb 53D10. Fixed and washed cells were incubated with Cy5-labeled DNA probes specific for green fluorescent protein (GFP, negative control) or IFNγ, and then analyzed by FC. The frequencies of cytokine producers are shown in FIG. 4. More than a 2-fold increase in expression of IFNγ mRNA occurred in Ag-specific T cells of LTBI donor in the presence of anti-CD28 mAb. Non-specific hybridization with control GFP and HIV-1 GAG probes was not affected by CD28 co-stimulation. Example 9 illustrates a distinct strategy to amplify the signal, namely, by providing co-stimulatory signals to TCR specifically engaged by cognate Ag. Furthermore, since co-stimulation mediated by anti-CD28 mAb did not affect non-specific binding of control probes (GFP and HIV-1 GAG), co-stimulation can be used to increase the strength of a specific signal over background noise.

Several strategies are particularly helpful for optimization of assays according to this invention. They include the following:

i) Kinetics of Signal Detection.

Kinetics of mRNA analyte expression for each of the RNA targets (for example, mRNA for cytokines IL-2, IFNγ and TNFα) are determined with cells of a given source (for example, Ficoll-separated PBMC obtained from blood samples of LTBI+ and LTBI− donors) that are stimulated with an appropriate inducer or inducers (for example, a mixture of Ags (for example, ESAT6- and CFP10-derived peptides (T-SPOT.TB, Oxford Immunotec, UK) present at 5 μg/ml), as in Example 8. Expression of each cytokine is then assayed separately in Ag-stimulated. PBMC at various times (for example, 30 min, 2, 4, 8, 12, and 16 h). Particularly for cytokines, PBMC non-specifically stimulated with anti-CD3 mAb and phytohemagglutinin (PHA) serve as positive controls of stimulation. In an assay for analysis of specific PBMC response, a preferred method for measuring non-specific response utilizes and compares PBMC from populations that comprise individuals known to be unaffected and affected by the condition for which the specific response is measured.

ii) Dose Response.

Dose response to Ag stimulation can be similarly determined (for example, for each of the three cytokines) by varying concentrations (1 to 20 μg/ml) of ESAT6 and CFP10 peptide mixture at the optimal signal detection time point. Additionally, non-specific stimulation (anti-CD3+PHA) of PBMC from any donors can be optimized by testing various concentrations, separate and in combination, to provide a standard positive control for a particular assay. In an assay for analysis of specific PBMC response, a preferred method for measuring non-specific response utilizes and compares PBMC from populations that comprise individuals known to be unaffected and affected by the condition for which specific response will be measured.

iii) Signal to Noise Amplification.

Probe sets with more or more highly intense fluorophores can be designed. This goal will also be achieved through several improvement strategies. Additionally, probe labeling methods can be modified as described earlier. Hybridization stringency can be optimized empirically in order to increase signal/noise ratio.

A distinct strategy to amplify the signal is by providing co-stimulatory signals to TCR specifically engaged by cognate Ag, as described in connection with Example 9. Conditions for co-stimulation can be optimized as outlined above for kinetics and dose-response to achieve robust detection of multiple RNAs (for example, three cytokines) by assay described herein.

iv) Threshold Determination.

Initial evaluation of a threshold for RNA detection can serve to standardize the optimized assay, although the clinically accepted threshold for a particular assay can be determined at a later point of commercial assay development. In the research phase, one can evaluate the threshold of RNA detection (cytokine expression) in Ag-specific T cells obtained from donors and stimulated ex vivo. The Ag-specific responses can be calculated by subtracting signal from unstimulated PBMC from the values obtained with Ag-stimulated PBMC at optimal time and Ag dose. For each cytokine read-out, a ROC (receiver operating characteristic) curve is estimated to show the trade-off between sensitivity and specificity at various cut points (including the mean of the donors plus two standard deviations), and optimal threshold values are selected to identify Ag-specific responses.

v) Reduction of Blood Volume and Processing Time.

Currently, 2×10⁶ cells/parameter (for example, each cytokine) that corresponds, on average, to 0.8 ml of blood, are analyzed. By introducing red blood cell lysis of the whole blood instead of Ficol separation in PBMC preparation, and further blood processing modifications such as vacuum-driven sedimentation (96-well format) instead of centrifugation, one can reduce sample volumes and shorten the time of assay. Preliminary experiments indicate that the hybridization time can be reduced to 2 hours (hrs) without a significant loss of signal.

Assay development can proceed by conventional methods. For example, PBMC from three donor groups (active TB, LTBI, and non-infected controls) can be stimulated with HLA-A2- and HLA-DR4-based aAPC loaded with ESAT6 and CFP10 peptides, as in Example 4. Each assay can test a panel of 4 markers of response to stimulation (assayed together based on expression properties) chosen from a group including but not limited to genes shown in Table 1 using 4-color FC for analysis. This allows all targets to be assessed for each donor. Markers selected in initial tests can be re-tested in combinations chosen to group together genes expressed at similar levels in each group. Statistical analysis can be performed as described in Example 4. Where desired, one can also measure expression of targets induced by conventional APC stimulation to confirm the ability of aAPC to improve detection sensitivity for multiple analytes within single cells. Further, an assay can also be tested by fluorescence microscopy. Statistical analysis can be performed as described in Example 5.

The experiment described in Example 10 demonstrates that the same methodology, which is referred to as “FISH-Flow” used for detection of cytokine mRNAs expression in T cells, can be successfully utilized in other types of cells, including but not limited to primary macrophages. Moreover, the experiment demonstrates that responses to different stimuli can be distinguished in primary macrophages by monitoring expression of a single gene at different times.

The results shown in FIG. 5 show that background expression assessed by a GFP negative control remained constant but negligible (approx. 0.1-0.2% of gated cells). Treatment with a mixture of lipopolysaccharide (LPS) and IFNγ or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyklysyl)3-lysine (Pam₃CSK₄) readily induced expression of TNFα transcripts in these cultured macrophages to levels of 6 to 42% of all cells being positive. Robust expression of TNFα transcripts is expected in activated macrophages and contrasted with a modest (less than 2%) expression of IFNγ mRNA, which was unaffected by the choice of stimuli and/or length of stimulation and thus may reflect an unusually high background in these cells rather than the real expression profile. The kinetics of TNFα mRNA expression induced by LPS and IFNγ appears to be different from that induced by Pam₃CSK₄, with the former stimulation resulting in a sustained prolonged elevation of mRNA levels. In contrast, stimulation of the same mRNA by Pam₃CSK₄ was significantly less persistent.

Example 11 demonstrates identification of temporal profiles of gene expression for cytokines, chemokines, and chemokine receptors, which are markers of T cell activation, in stimulated human T cells, comparing flow cytometry results obtained using FISH probes specific for the indicated mRNAs (FIG. 6A) or using mAbs recognizing their protein products (FIG. 6B). The frequencies of cytokine producers by FISH-Flow (FIG. 6A) showed that expression of IL-2, IFNγ, TNFα, CCL3, CCR7 and cFos mRNAs in non-stimulated cells is either negligible (control GFP, IL-2, IFNγ and CCR7) or very low (TNFα, CCL3 and cFos), occurring in <1% of gated cells. Unexpectedly, induced expression of these mRNAs seems to peak at 2 hours after stimulation and then to decline through 1 day and 3 day time points. This is in contrast to the parallel data obtained with the same cells (in the presence of 1 μg/ml brefeldin A) by conventional mAb immunostaining and flow cytometry. Here, non-stimulated cells clearly show basal levels (1-3%) of protein expression detected for at least some markers such as IL-2, IFNγ and TNFα in comparison to a negative control—isotype-matched (Iso) mAb. Furthermore, all the targets are induced at 2 hours and remain elevated in the 3 day time period examined. Thus, no distinct temporal profiles could be detected with mAbs that bind to the cytokines. Example 11 demonstrates differences in kinetics that provide superior profile discrimination with FISH-Flow versus conventional mAb staining and flow cytometry and suggests that the former method provides greatly improved sensitivity for detection of activation.

In order to fully define any cell type functional signatures (i.e., capability to perform specialized biological functions), knowing expression of all other mRNAs in activated cells vs. non-activated (non-responding) cells may be important. The study reported in Example 12 demonstrates that FISH-Flow enables labeling of cells that express one or multiple targets in response to stimulation, and that such responders could be isolated from the whole cellular population for the purpose of interrogating their gene expression profiles.

Stimulated human T cells were incubated either with Cy5-labeled DNA probes complementary to RNA specific for GFP and analyzed by FC as a control or with a mixture of several FISH probe sets (Table 3) reactive with IL-2, IFNγ, TNFα, and CCL3 mRNAs, all probes singly-labeled with Cy5. Results obtained with the GFP probes are shown in FIG. 7A. Results obtained with the probes mixture are shown in FIG. 7B. After gating for the lymphocyte population (left panel in each figure), conservative additional gating was applied to separate the analyzed lymphocytes into two distinct populations (right panel in each figure) that would distinguish cells expressing IFNγ (IFN+) from the non-expressing cells (IFN−). The gating set up for GFP-probed cells (FIG. 7A) defined two populations, 0.1% in the IFN+ population and 71.2% in the IFN− population, out of all analyzed lymphocytes. The IL-2:IFNγ:TNFα:CCL3-probed cells (FIG. 7B) were 10.2% IFN+ and 33.7% IFN− with the same gating. The latter FISH-probed cells (FIG. 7B) were separated into the positive and negative populations by fluorescence-activated cell sorting (FACS) in order to perform gene expression analysis on isolated activated and non-responding cells. The sorted cell populations were analyzed to demonstrate distinct gene expression profiles consistent with their reactivity with specific FISH probes. Expression of mRNAs encoded by two genes, IFNγ and “housekeeping” GAPDH (control), was chosen to exemplify an embodiment of this aspect of the invention. The sorted cell populations were subjected to RT-PCR using primers specific to one of the two genes. Quantitative measurement of amplified product was made by including SYBR Green dye in the amplification reaction mixture. Results, shown in FIG. 8A, demonstrate that IFNγ-specific primers detect a greater abundance of target in IFN+ cells that were identified with probes for IL-2, IFNγ, TNFα and CCL3 than in IFN− cells that were minimally labeled with those probes. Amplification of a control GAPDH signal that is unaltered by activation status is expected to be similar in both activated and non-responding cells. Slightly reduced amplification seen with GAPDH-specific primers in activated cells reflects the fact that this sorted population is significantly smaller, and, therefore, could yield less mRNA.

These results (confirmed by size analysis, FIG. 8B) demonstrate that gene expression may be further analyzed in desired subpopulations of cells identified using the FISH-flow platform of this invention by applying methods known to those well-versed in the art for sorting and obtaining subpopulations of cells, and methods for gene expression measurement such as RT-PCR or transcriptomic analysis. The subpopulation that can be identified and studied may contain one cell or more than one cell, including but not limited to 10, 100, 1000, 10,000, 100,000, or more cells.

Examples Example 1 Demonstration of Induction and Probe Sets

This example demonstrates the use of artificial stimulation of cells to produce RNA, in this case mRNA, and the use of sets of singly labeled fluorescent probes to hybridize to expressed RNAs.

For this example, the technique of smFISH was used to visualize spots corresponding to individual molecules of mRNAs bound to the probes. See Raj et al., 2010, Methods in Enzymology 472:365-386; and Raj et al., 2008, Nature Methods 5: 877-879. The probe hybridization conditions and cell washing conditions are described in these references. The procedure departed in one important way. In order to avoid the loss of cells during washing steps 0.5% fetal bovine serum was included in the washing solutions. A set of nucleic acid probes, here DNA probes, were selected for TNFαmRNA, a key target for analysis of activated T cells and macrophages, and tested differentiated THP-1 cells stimulated with irradiated M. tuberculosis. Also included were a second set of DNA probes, labeled with a fluorophore of different color and designed to bind to ACSL1 mRNA (a lipid metabolism gene). The latter gene was also expected to be induced by the stimulus. The probe set specific for human TNFα comprised 48 probes, each about 20 nucleotides long and terminally labeled with a tetramethylrhodamine fluorophore. The probe set specific for human ACSL1 gene comprised 48 probes, each about 20 nucleotides long and labeled with an optically distinguishable fluorophore, namely, alexafluor 594. The sequences of the probes are listed in the sequence listing at the end of this description (Table 3). Although the probes listed in each set are suitable for detection of the target mRNA, alternative probes selected from different regions of the target RNAs can also be used as described in Raj et al., 2010, Methods in Enzymology 472:365-386; and Raj et al., 2008, Nature Methods 5: 877-879. As discussed above, spots corresponding to both mRNAs in the cells were detected. The number of TNFα RNA molecules increased from 0.24 per cell without induction to 14 per cell, on average, following stimulation.

Example 2 Use of FC to Detect Cells Expressing a Gene

In this experiment assays were carried out to detect HIV GAG mRNA in cell cultures expressing a lentiviral construct.

Briefly, 293T cells were transfected with three plasmids that together allow expression of a recombinant lentiviral construct and hybridized with probes specific for GAG region of HIV mRNA (GenBank Accession #AY835771.1). The sequences of the probes are listed at the end of this specification (Table 3). The probes were hybridized and the cells were washed according to the procedures described in Raj et al., 2010, Methods in Enzymology 472:365-386; and Raj et al., 2008, Nature Methods 5: 877-879, which are incorporated by reference herein in their entireties. Again, to avoid the loss of cells during washing steps, 0.5% fetal bovine serum was included in the washing solutions. FC was then performed to detect cells expressing the construct. A gate was established based on the fluorescence of cells that were not transfected. In cells that were transfected, about 25% of the cells expressed the construct as shown by signal of greater intensity than the level set by the gate. FC results are presented in FIG. 1, which shows two graphs of fluorescence intensity (“a.u.”) versus forward scatter (“FSC-A”). The graph on the left (Lentivirus−) shows cells that were not transfected. The graph on the right (Lentivirus+) shows cells that were transfected with three plasmids from a lentiviral packaging system.

Example 3 Detection of Cytokine Gene Expression Stimulated by M-TB Derived Peptides

PBMC are obtained from uninfected, asymptomatic latent infection (LTBI), and active TB donors. T cells are activated by addition of the peptide mixture used in the T-SPOT.TB IFNγ Release Assay (IGRA) (Oxford Immunotec, Marlborough, Mass.) according to the manufacturer's instructions, which is derived from immunodominant mycobacterial Ags Rv3875 (ESAT6) and Rv3874 (CFP10). At various times (4 to 120 h), stimulated cells are fixed and permeabilized and then hybridized with one or more of the probe sets described below. The cells are analyzed by FC to determine the frequency of cells that express the targets, individually and in combination, using available filter sets that distinguish among the fluorophores used to label the probes.

The probe set for TNFα is the probe set specified in Example 1. Two additional probe sets, one for TL-2 and one for IFNγ are prepared, each having 50 probes (Ensembl Sequence IDs ENST00000226730 and ENST00000229135 respectively). The probes in all three sets are linear (random coil) oligonucleotides 15-25 nucleotides long, labeled with a single fluorophore. The three fluorophores used for the three sets are optically distinguishable. These three cytokine genes are known markers for T-cell activation and proliferation that distinguish T-cell subtypes. The target transcripts are sufficiently long to permit the use of at least 50 probes for each.

To detect marker expression in a small fraction (≦0.01%) of cells by FC, results for 1×10⁶ events are collected. To determine statistical significance for differences between donor groups, a threshold for separation of non-responsive from responsive cells is determined by ROC analysis (see, e.g., Zweig et al., 1993, Clinical Chemistry 39 (8): 561-577 and Pepe, 2003, The statistical evaluation of medical tests for classification and prediction. New York, N.Y.: Oxford), and the frequency of positive cells is determined. A mixed-model ANOVA can be used to analyze the data. Data is transformed (e.g., log-transformation), prior to analysis. In cases where a simple transformation is insufficient to address ANOVA assumptions, a non-parametric analog of the statistical approach is applied.

A detectable frequency of expression for all three markers is found in the activated cells. The frequencies of cells that express different combinations of the markers is the signature of the disease.

Example 4 Assay for M. Tuberculosis-Specific Effector T Cells

This example describes assays for detecting M. Tuberculosis-specific effector T cells.

a. Detection of CD8+ T Effectors.

TB patients are HLA typed by FC detection of PBMC immunostained with allele-specific mAbs BB7.2 and 0222, or by PCR confirmation. HLA-A*0201-based aAPC is loaded with known individual HLA-A*0201 epitopes (peptides) derived from Rv1886c, Rv3874 and Rv3875.

These peptides are derived from immunodominant Ags of M. tuberculosis. PBMC are isolated from HLA-A2-typed TB patients and are stimulated with the Ag-loaded, A2-based aAPC at 1:1 ratio under standard culture conditions. Alternatively, isolated PBMC are incubated with added peptides for T cell stimulation by endogenous APC. At various times from 4 to 120 h, cells are fixed and permeabilized and incubated with specific probes for IL-2, IFNγ, and TNFα. Negative controls include PBMC from healthy, non-infected (IGRA-negative) donors stimulated with the above-described Ag-loaded aAPC and PBMC from positive donors stimulated with aAPC loaded with the irrelevant melanoma-specific Mart 1 peptide (26-35 epitope) and differentiation Ag gp100 peptide (44-59 epitope). Positive controls include PBMC non-specifically stimulated with anti-CD3 mAb+PHA.

b. Detection of CD4+ T Effectors.

First HLA-DRB1*04-based aAPC is constructed using the methodology described above for the assembly of HLA-A*0201-based aAPC. A major aspect of this approach is the expression of a soluble form of HLA-DR4 molecules, which requires pairing of the class II a and b polypeptides during biosynthesis. To facilitate subunit pairing of soluble analogs of MHC class II molecules, the IgG molecule is used as a molecular scaffold: it is divalent and can be readily modified to accommodate a wide variety of protein domains. Modified a and b chains are cloned in a dual promoter baculovirus expression vector. The cells infected with this vector secrete approximately 0.5-2.0 mg/ml of the soluble HLA-DR4-Ig-like material. To assemble soluble MHC class II HLA-DR4-Ig complexes using a generic cloning vector pZig, DNA encoding class II extracellular domains of α and β chains of DR4 proteins is ligated to DNA encoding Ig heavy and light chains using endonuclease restriction sites; and the chimeras are ligated sequentially to a modified pAcUW51 vector. The yield, purity, and integrity of secreted proteins is confirmed by biochemical analysis (SDS-PAGE and Western blotting) with HLA-DR4-specific mAb 0222HA (One Lambda). Purified soluble HLA-DR4-Ig proteins and anti-CD28 mAb at 1:1 ratio are conjugated to magnetic beads (M-450 Epoxy Dynabeads, Dynal Invitrogen).

Known peptides derived from Rv1886c, Rv3874 and Rv3875 and restricted by the HLA-DRB1*04 allele (Table 1) are loaded on HLA-DR4-based aAPC and used to stimulate PBMC derived from non-infected, LTBI and active TB donors. Alternatively, isolated PBMC are incubated with added peptides for T cell stimulation by endogenous APC. After stimulation for various times from 4 to 72 h, cells are probed for IL-2, IFNγ, and TNFα mRNA and analyzed by FC. Negative and positive controls are as described above for CD8+ T cells.

TABLE 1 HLA-A*0201- and HLA-DRB1*04-restricted epitopes M. tb HLA-A2 epitope HLA-DR4 epitope protein (SEQ ID NO.) (SEQ ID NO.) Rv1886c FIYAGSLSAL (1) PVEYLQYPSPSMGRD (4) KLVANNTRL (2) LPVEYLQVPSPSMGR* (5) GLAGGAATA (3) PQQFIYAGSLSALLD* (6) Rv1986 ALGISLTV* (7) AGRLRGLFTNPGSWR (10) FLACFTLIAA* (8) VGFLACFTLIAAIGA* (11) FLIGYGLLA* (9) LDTVVLLGALANEHS* (12) Rv2220 GLLHHAPSL (13) -*** SLWKDGAPL (14) Rv2659c RKAAGRPDLRV* (15) PDPYQAFVLMAAWLA* (18) KLLDNHILA* (16) TMRAHSYSLLRAIMQ* (19) FVLMAAWLA* (17) RAHYRKLLDNHILAT* (20) Rv2780 VLMGGVPGVE (21) -*** LLDSGTTSI (22) Rv3407 ARVEAGEELGV* (23) AIGIRELRQHASRYL* (26) ALIESGVLIPA* (24) RLVARLIPVQAAERS* (27) RLVARLIPV* (25) KRTLSDVLNEMRDEQ* (28) Rv3874 SGDLKTQIDQV* (29) IRQAGVQYSR** (30) IRQAGVQYSR** (30) EISTNIRQA (31) Rv3875 AMASTEGNV (32) FAGIEAAASAIQGNV (34) LLDEGKQSL (33) SAIQGNVTSIHSLLD* (35) *predicted using software availabe from the website of Immune Epitope Datase And Analysis Resource at immuneepitope.org. **this epitope is dually class I/II-restricted. ***not needed, only used with class I-based aAPC.

The methods of statistical analysis described in Example 3 are applied to these results. The multiple stimulation approaches in this example also require a mixed-model ANOVA for comparisons.

The foregoing test is able to detect both CD4+ and CD8+ effector T cells in the blood of TB patients based on stimulation with allele-specific HLA class I- or class II-restricted peptides. Activated effector T cells recognizing Rv1886c, Rv3874 and Rv3875 Ags and capable of producing at least a single cytokine, IFNγ or TNFα, are readily detectable. Co-production of two cytokines (IL-2+IFNγ+ and IL-2+ TNFα+) is less frequent in the terminally differentiated T cells that are the majority of effectors in the blood of patients with active TB.

Example 5 Single T Cell Cytokine Profiles Elicited by Infection Stage-Specific Ags

PBMC from active TB, LTBI, and non-infected donors are stimulated at various times (4 to 120 h) with HLA-A2- and HLA-DR4-based aAPC loaded with peptide mixtures derived from Rv1886c (active TB stage), and Rv1986, Rv2659c and Rv3407 (LTBI stage). Alternatively, isolated PBMC are incubated with added peptides for T cell stimulation by endogenous APC. The read-out is expression of IL-2, IFNγ and TNFα using three sets of about 50 singly labeled fluorescent probes and FC detection of numbers of expressing cells. To identify Ag candidates that may discriminate between disease states, one-way ANOVAs was used and a generous p-value (p=0.25) as a cutoff. Selected Ags are used to develop a model of responses that distinguishes donor groups using generalizations of logistic regression such as polytomous or cumulative logistic regression.

Stimulation with Rv1886c produces a characteristic, infection stage-associated T-cell cytokine profile: single, double, and triple producers with TB patients and mostly IL-2 producers with LTBI patients.

Example 6 Identification of Functional Effector and Memory Signatures Associated with Infection Stage in Single T Cells

Functional markers that are inducible by TCR activation can be measured by methods of this invention so as to accurately describe the main functional T-cell subsets. For this assay candidate markers that are inducible within 4-6 hours after TCR engagement and/or genes that reflect maturation stage and function of known T-cell subsets were selected. The resulting panel of activation and functional markers expressed in effector, effector memory and central memory T cells (Table 2) meets these considerations. The data in Table 2 was compiled from Zinkemagel R M. T cell activation. In: Mak T W, Saunders M E, editors. New York: Elsevier; 2006. p. 373-401, and from Doherty P. T cell differentiation and effector function. In: Mak T W, Saunders M E, editors. New York: Elsevier; 2006. p. 403-432, and references therein.

TABLE 2 Key markers defining T cell signatures* Marker Subset Function CD27 Naive, effector Proliferation, memory response CD40L Effector APC activation, primary response CD44high Effector, memory Memory response CD69high Effector, memory Activation, Th17 response CD107a CD8+ effector, CTL degranulation memory CD137 Effector CTL proliferation, IFN□ production IL-2 Effector, memory Proliferation, Th1 response IFNg Effector, memory Th1 response TNFa Effector, memory Th1 response MIP-1a Effector, memory Th1 response IL-2Ra Effector, memory Proliferation CCR1 Effector Homing CCR5 Effector Homing, activated memory CCR7 Central memory Homing, activated central memory CXCR4 Effector Homing, migration CXCR5 CD4+ memory Homing, migration, memory LPAM-1 Effector, memory Homing, CTL differentiation, memory VLA-4 Effector, memory Trafficking, adhesion, memory Bcl-2 Memory Anti-apoptosis, cell survival Bcl-xL CD8+ memory Anti-apoptosis, cell survival Perforin CD8+ effector Cytotoxicity Granzyme B CD8+ effector Cytotoxicity

The use of aAPC loaded with peptides (as in Example 3 or 4) to provide stimulation via TCR and co-stimulation via CD28, or alternatively, anti-CD28 mAb combined with peptides (as in Example 3 or 4) for stimulation by endogenous APC, rapidly increases expression of key chemokines and cytokines (including but not limited to MIP-1α, IFNγ, and TNFα) and other co-stimulatory molecules (including but not limited to CD40L, CD137), which in turn further increases responsiveness. The positive feedback augments responses in the time frame utilized for detection and increases the sensitivity of FC results for multi-parameter analysis. The combinations of selected markers are chosen to enable reproducible identification and characterization of individual effector and memory cells and their quantitative analysis in the peripheral blood of donor groups. They serve as T-cell functional signatures. Four-color FC is used so that the signature(s) identified are suitable for analysis with instruments commonly used in clinical settings.

High donor reproducibility is found for data obtained with PBMC stimulated with the commercial mixture of ESAT6 and CFP10 peptides and HLA-A2- or HLA-DR4-based aAPC. The data obtained with this mixture of peptides are very accurate with conventional IGRA based on endogenous APC. The robust stimulation provided by aAPC extends to induction of multiple targets, as described above for standard cytokine targets. Cells expressing multiple markers are also detected at higher frequency than would be the case for conventional APC stimulation. The method of this example permits identification of marker sets that serve as signatures of distinct T cells that are present at different stages of infection, here TB infection.

Example 7 Simultaneous Detection of Two Cytokine mRNAs in Stimulated PBMC

This example shows that it is possible to detect a very small population of fluorescent cells. Briefly, CFSE-labeled PMBC was serially diluted into MITOTRACKER (Invitrogen)-labeled PBMC. It was found that the assay was able to detect the former at a frequency of 0.0013%, demonstrating sufficient sensitivity.

For demonstrating the detection of IL-2, IFNγ and TNFα mRNA, PBMCs were stimulated non-specifically via TCR with anti-CD3 mAb and PHA that is known to induce expression of various cytokines in these cells. After stimulation, the cells were fixed and probed with pairwise combinations of probe sets against IL-2 and IFNγ, IL-2 and TNFα, and also IFNγ and TNFα. One probe set in each pair was labeled with Cy5 fluorophore and the other was labeled with TMR fluorophore. Specifically, PBMC were stimulated with anti-CD3 mAb OKT3 and PHA (both at 1 mg/ml) for 5 days, and then fixed with 4% paraformaldehyde and 70% ethanol. Fixed and washed cells were labeled by incubating with Cy5- or TMR-labeled RNA probes specific for IL-2 and IFNγ, washed again and analyzed by two-color FC (LSRII, Becton Dickinson). Results are shown in FIG. 2. The four plots in FIG. 2 are, from left to right, unstained/mock-hybridized control; single cytokine, IFNγ-Cy5-labeled cells; single cytokine, IFNγ-TMR-labeled cells; and double cytokine (IL-2, Cy5-labeled and IFNγ, TMR-labeled)-labeled cells. Gating shows cell populations expressing one or two cytokines and their frequencies.

Example 8 Detection of Cytokine mRNAs in M. Tuberuclosis-Specific Cells Stimulated Ex Vivo

In this example, a method according to this invention was applied for the detection of M. tuberculosis-specific cells stimulated ex vivo. Circulating M. tuberculosis-specific cells are typically present at low frequencies in persons with latent TB infection (LTBI). Blood from an LTBI donor was obtained and stimulated for 5 hr with a mixture of peptides derived from M. tuberculosis Ag ESAT6 and CFP10 (commonly used in T-SPOT.TB test). PBMC were incubated for 5 h either in medium alone or with a mixture of ESAT6- and CFP10-derived peptides (T-SPOT.TB, Oxford Immunotec, UK) at 5 μg/ml. Fixed and washed cells were incubated with Cy5-labeled DNA probes complementary to RNA specific for GFP as a negative control, or for IL-2 and TNFα, and then analyzed by FC. The frequencies of cytokine producers are shown in FIG. 3, wherein the three upper plots are for PBMC incubated in medium alone, and the three lower plots are for PBMC incubated in medium containing the Ag mixture.

Example 9 IFNγ Detection in Co-Stimulated PBMC of LTBI Donor

PBMC were incubated for 5 h with a mixture of ESAT6- and CFP10-derived peptides (T-SPOT.TB, Oxford Immunotec, UK) at 5 μg/ml, with or without anti-CD28 mAb 53D10. Fixed and washed cells were incubated with Cy5-labeled DNA probes specific for GFP mRNA (GFP, negative control) (GFP sequence refers to pTREd2EGFP (Invitrogen) or IFNγ mRNA and then analyzed by FC. The frequencies of cytokine producers are shown in FIG. 4. Non-specific staining of control GFP was not affected by CD28 co-stimulation.

Example 10 Cytokine Induction in Activated Macrophages

This example illustrates the use of artificial stimulation of non-T cells, in this case, human macrophages obtained from peripheral blood by adherence to plastic and cultured for 2 weeks under standard conditions (RPMI 1640 media supplemented with 10% fetal bovine serum and antibiotics).

Briefly, activation was carried out with a mixture of lipopolysaccharide (LPS) (100 ng/ml) and IFNγ (20 ng/ml) or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine (Pam₃CSK₄) (100 ng/ml) for 4 hours or 1 day. Stimulated primary macrophages were removed from the plastic in 0.2-0.3% EDTA in PBS, fixed and permeabilized as described in Example 7, and then incubated with FISH probes specific for IFNγ and TNFα mRNAs (Table 3) and analyzed by flow cytometry. As in Example 8, fixed and washed cells were incubated with Cy5-labeled DNA probes complementary to RNA specific for GFP (Table 3) and analyzed by FC as a negative control. The frequencies of cells detected with the probes by FISH-flow analysis, described above, for instance in example 2, are shown in FIG. 5, wherein the top row is results with the GFP probes, the middle row is for the IFNγ probes, and the bottom row is for the TNFα probes. The first column in each row is stimulation with LPS and IFNγ for 4 hours, the second is stimulation with LPS and IFNγ for 1 day, the third is stimulation with Pam₃CSK₄ for 4 hours, and the fourth is stimulation with Pam₃CSK₄ for 1 day.

Example 11 Expression Kinetics of Cytokine and Activation Markers in Activated T Cells

This example demonstrates identification of temporal profiles of gene expression for cytokines, chemokines, and chemokine receptors, which are markers of T cell activation, in response to stimulation with PMA (25 ng/ml) and ionomycin (350 ng/ml) of human T cells obtained from peripheral blood on Ficoll gradients. More specifically, after various periods of stimulation (2 hours, 1 day, 3 days), cells were fixed and permeabilized as described in Example 7, and then were either incubated with FISH probes specific for the indicated mRNAs (Table 3) or with mAbs recognizing their protein products followed by flow cytometric analysis. The frequencies of cells detected with the probes by FISH-flow analysis, described above, for instance in example 2, are presented in FIG. 6A, wherein the top row is results without stimulation and the subsequent rows are results after stimulation for 2 hours, 1 day and 3 days, respectively. The first column in each row is results using GFP probes; the second column, using IL-2 probes; the third column, using IFNγ probes; the fourth column, using TNFα probes; the fifth column, using CCL3 probes; the sixth column, using CCR7 probes, and the final column, using cFos probes. The top row is for the GFP probes, the middle row is for the IFNγ probes, and the bottom row is for the TNFα probes. The frequencies of cells detected with the mAbs by FC are presented in FIG. 6B, wherein the top row is results without stimulation and the subsequent rows are results after stimulation for 2 hours, 1 day and 3 days, respectively. The first column in each row is results using a negative control—isotype-matched (Iso) mAb; the second column, using mAb recognizing IL-2; the third column, using mAb recognizing IFNγ; the fourth column, using mAb recognizing TNFα.

Example 12 Separation and Analysis of Activated Fish-Probed T Cells into Cytokine-Expressing and Non-Expressing Populations

This example demonstrates that the above-described FISH-Flow enables labeling of cells that express one or multiple targets in response to stimulation, and that such responders could be isolated from the whole cellular population for the purpose of interrogating their gene expression profiles.

Part I: Separation into Populations

Human T cells obtained from peripheral blood on Ficoll gradients and globally stimulated with PMA (25 ng/ml) and ionomycin (350 ng/ml) for 2 hours were incubated either with Cy5-labeled DNA probes complementary to RNA specific for GFP and analyzed by FC as a control or with a mixture of several FISH probe sets (Table 3) reactive with IL-2, IFNγ, TNFα, and CCL3 mRNAs, all probes singly-labeled with Cy5. The frequencies of the cells detected with the probes by FISH-Flow analysis, described above, for instance in example 2, are presented in FIG. 7A and FIG. 7B. Results obtained with the GFP probes are shown in FIG. 7A. Results obtained with the probes mixture are shown in FIG. 7B. After gating for the lymphocyte population based on FSC and SSC (left panel in each figure), conservative additional gating was applied to separate the analyzed lymphocytes into two distinct populations (right panel in each figure)—cytokine positive (denoted IFN+) and negative (denoted IFN−). The gating set up for GFP-probed cells (FIG. 7A) defined two populations, 0.1% IFN+ and 71.2% IFN−, out of all analyzed lymphocytes. The IL-2:IFNγ:TNFα:CCL3-probed (FIG. 7B) populations were 10.2% IFN+ and 33.7% IFN− with the same gating. The latter FISH-probed cells (FIG. 7B) were separated into the positive and negative populations by FACS in order to perform gene expression analysis on isolated cytokine-expressing and non-expressing cells as described below. This approach was also successful for stimulated T cells labeled with only an IFNγ-specific FISH probe that were distinguished and sorted into IFN+ and IFN-subpopulations.

Part II: Cytokine Gene Expression Analysis of FISH-Probed and Sorted into Activated and Non-Responding T Cell Populations.

Activated and non-responding T cells prepared and sorted into separate populations as described in Part I were analyzed to demonstrate distinct gene expression profiles consistent with their reactivity with specific FISH probes. Expression of mRNAs encoded by two genes, IFNγ and “housekeeping” GAPDH (control), was chosen to exemplify feasibility of this goal. RNA isolated from the sorted cell populations by standard methods was subjected to RT-PCR (reverse transcription followed by amplification of the resulting cDNA by the polymerase chain reaction) using a QIAGEN OneStep RT-PCR Kit following the protocol described by the vendor, and using primers specific to one of the two genes. Primer sequences for the amplification reactions were as follows:

IFNγ forward primer: (SEQ ID NO.: 36) 5′ GAGCATCCAAAAGAGTGTGGAG IFNγ reverse primer: (SEQ ID NO.: 37) 5′ TTCATGTATTGCTTTGCGTTGG GAPDH forward primer: (SEQ ID NO.: 38) 5′ CCAATATGATTCCACCCATGGC GAPDH reverse primer: (SEQ ID NO.: 39) 5′ TCCTGGAAGATGGTGATGGGAT Following the RT incubation and initial denaturation for 15 min. at 94° C., the thermal cycling steps were: 0.5 min at 94° C., 0.5 min at 55° C., 1 min at 72° C. Forty cycles were performed. Detection of amplified products was by SYBR Green dye. Threshold cycles for the amplification reactions are shown in FIG. 8A. To validate the amplification results and exclude a possibility of artifact, the size of identified amplicons was verified with IFNγ- and GAPDH-specific primers by gel electrophoresis. Results are shown in FIG. 8B.

TABLE 3 Probe Sequences SEQ SEQ ID ID NO NO IFNγ probes  40 atcagaacaatgtgctgcac  64 tctaatagctgatcttcaga  41 ccaaaggacttaactgatct  65 tcttgtatcaagctgatcag  42 gccaaagaagttgaaatcag  66 tcatcgtttccgagagaatt  43 gagctgaaaagccaagatat  67 caagagaacccaaaacgatg  44 tatgggtcctggcagtaaca  68 aaggttttctgcttctttta  45 gacctgcattaaaatatttc  69 ccattatccgctacatctga  46 caaaatgcctaagaaaagag  70 cactctcctctttccaattc  47 tggctctgcattatttttct  71 gtttgaagtaaaaggagaca  48 gctctggtcatctttaaagt  72 atggtctccacactcttttg  49 cttgacattcatgtcttcct  73 ctcgtttctttttgttgcta  50 ttagtcagcttttcgaagtc  74 gacattcaagtcagttaccg  51 gttcatgtattgctttgcgt  75 agttcagccatcacttggat  52 ttcgcttccctgttttagct  76 cgaaacagcatctgactcct  53 attactgggatgctcttcga  77 caaatattgcaggcaggaca  54 ccccatataaataatgttaa  78 cttatttgattgatgagtct  55 cattacacaaaagttgctat  79 gacagtcacaggatatagga  56 cagaaaacaaaggattaagt  80 cacatagccttgcctaatta  57 ccctgagataaagccttgta  81 ttaggttggctgcctagttg  58 aaacacacaacccatgggat  82 gttcattgtatcatcaagtg  59 ctggatagtatcacttcact  83 catattttcaaaccggcagt  60 aagcactggctcagattgca  84 agttctgtctgacatgccat  61 tcagggtcacctgacacatt  85 ctcctgagatgctatgtttt  62 ttggaagcaccaggcatgaa  86 cagtcacagttgtcaacaat  63 tgagttactttccatttggg  87 gtgaacttacactttattca IL-2 Probes  88 tagcccacacttaggtgata 112 gtgaaatccctctttgttac  89 agactgactgaatggatgta 113 ggaatttctttaaaccccca  90 ttcctcttctgatgactctt 114 gaaaaaacattaccttcatt  91 tttcaaagactttacctgtc 115 tgttttacatattacacata  92 aatattatgggggtgtcaaa 116 ttatactgttaattctggaa  93 ctcttgaacaagagatgcaa 117 attaaagagagtgataggga  94 ttgaggttactgtgagtagt 118 tcctgtacattgtggcagga  95 caatgcaagacaggagttgc 119 tgacaagtgcaagacttagt  96 ttgaagtaggtgcactgttt 120 gctgtgttttctttgtagaa  97 gcagtaaatgctccagttgt 121 tcaaaatcatctgtaaatcc  98 tcttgtaattattaattcca 122 gcatcctggtgagtttggga  99 gcatgtaaaacttaaatgtg 123 tcagttctgtggccttcttg 100 cttctagacactgaagatgt 124 cctccagaggtttgagttct 101 tttgagctaaatttagcact 125 gtcttaagtgaaagtttttg 102 tattgctgattaagtccctg 126 gttccagaactattacgttg 103 atgttgtttcagatcccttt 127 catcagcatattcacacatg 104 attctacaatggttgctgtc 128 aggtaatccatctgttcaga 105 gttgagatgatgctttgaca 129 gcacttaattatcaagtcag 106 ctgatatgttttaagtggga 130 atatttaaataaatagaagg 107 caacaataaatataaaattt 131 ataggtagcaaaccatacat 108 agattaagaataatagttac 132 agatccatatttatagtttt 109 gggcttacaaaaagaatcat 133 gaaaccattttagagcccct 110 aatattttgggataaataag 134 ctatatttaacattcaacat 111 actaaccaatctacatagat 135 tcaaatttattaaatagttt TNFα probes 136 tcctctgctgtccttgctga 160 agttgcttctctccctctta 137 tctgagggttgttttcaggg 161 atgtgagaggaagagaacct 138 tgtcctttccaggggagaga 162 gatcatgctttcagtgctca 139 aagaggctgaggaacaagca 163 aaagtgcagcaggcagaaga 140 tgattagagagaggtccctg 164 agaagatgatctgactgcct 141 tgggctacaggcttgtcact 165 agcttgagggtttgctacaa 142 tggttatctctcagctccac 166 tgaggtacaggccctctgat 143 ttgaagaggacctgggagta 167 ttgaccttggtctggtagga 144 gctcttgatggcagagagga 168 agatagatgggctcatacca 145 acccttctccagctggaaga 169 attgatctcagcgctgagtc 146 tcggcaaagtcgagatagtc 170 atgatcccaaagtagacctg 147 tttgggaaggttggatgttc 171 taataaagggattggggcag 148 agaggttgagggtgtctgaa 172 cccaattctctttttgagcc 149 ttctaagcttgggttccgac 173 gtggtggtcttgttgcttaa 150 attcctgaatcccaggtttc 174 tagtggttgccagcacttca 151 tggaggccccagtttgaatt 175 aaagctgtaggccccagtga 152 tctccagattccagatgtca 176 attctggccagaaccaaagg 153 taggtgaggtcttctcaagt 177 ctaaggtccacttgtgtcaa 154 aaacatctggagagaggaag 178 tccgtgtctcaaggaagtct 155 ataaatagagggagctggct 179 ccggtctcccaaataaatac 156 acattgggtcccccaggata 180 aaaacatgtctgagccaagg 157 ttgttcagctccgttttcac 181 atcaaaagaaggcacagagg 158 ggtcaccaaatcagcattgt 182 aggctcagcaatgagtgaca 159 attacagacacaactcccct 183 ttctcgccactgaatagtag ACSL1 probes 184 tgcaatgtgatgcctttgac 208 gaaggccattgtcgatagaa 185 ttcgccttcattgttggagt 209 tgaaatagttccgcagctct 186 tagaggtcatctatctgcga 210 acactaaaccttgatagtgg 187 ccatttcctctgagctttct 211 gagaagagattgtggaactg 188 aacaacatgaaggccatcag 212 ccctacacttgctgtattca 189 aagtcaaacacgaacgcttc 213 tatgagaagaaccccgaatg 190 gtgttctctttcctctagca 214 aacatgaggtgactgtaagg 191 ggctcattttggaaagtgtg 215 gcacatttatagtatcccct 192 ctgaggaagtctcaaataac 216 gaaacagggagacagaagat 193 caaagtcctaaccccttgat 217 gtagcagacatctcagagat 194 agacagctgcagaatttgca 218 gcactgtactctttagagca 195 aaagggaacacttccctcta 219 gccaggacagttgttcttat 196 tggtccgcttgtgagattct 220 ggacagtagcagggatttaa 197 ttctgtgaatgcctgtgaga 221 cgtaacccttacgaatcaga 198 gactggagaagaacatgagt 222 atgctccaacagaaaccaca 199 atggttctgagttggatctg 223 atttgccagaggctccttat 200 aatccacgcgttctgatgag 224 agagcattctgccatgaaag 201 aacccctgaaaaaggatgga 225 cactatagaatctaaggcag 202 ctgatccaggtaactctttc 226 acagttattcaggcgtcaca 203 gtgagtggttcagtgaagat 227 ggcaagttaatcccaacatg 204 gaaatgcttgcctgagagtt 228 gtactcaagtatatactccc 205 ccaggaaatgaactttccag 229 caaatgtggcaaagactcac 206 ccccaaaacagctgctttaa 230 gccctgttgtgcttgtatat 207 gttaggctaccaagtagtgt 231 caaaaccccaaggtgacaaa CCL3 probes 232 ctgctgcccgtgtcctt 252 agaaaggactgaccact 233 ctcgagtgtcagcagag 253 gagcaggtgacggaatg 234 agtggagacctgcatga 254 gaggacagcaagggcag 235 gagagccatggtgcaga 255 tgcagagaactggttgc 236 cgtgtcagcagcaagtg 256 gtagctgaagcagcagg 237 gtggaatctgccgggag 257 agtcagctatgaaattc 238 ggctgctcgtctcaaag 258 caccgggcttggagcac 239 gcttggttaggaagatg 259 cacagacctgccggctt 240 cactcctcactggggtc 260 cgctgacatatttctgg 241 ctcaggcactcagetec 261 ctcgaagcttctggacc 242 ccaccgaggtcgctggg 262 tcaggctcctgctcctc 243 acacgcatgttcccaag 263 aagaggtagctgtggag 244 gcaacaaccagtccata 264 ccacagtgtggctgttt 245 tttaagttaagaagagt 265 agtataaataaattaaa 246 aaattacaaaaactaaa 266 acacactctgaaatcga 247 cagagcaaacaatcaca 267 aggggacaggggaactc 248 gttgtcaccagacgcgg 268 gctgatgacagccactc 249 gccatgactgcctacac 269 tcagtctggtggctttg 250 gcatccgatacacattt 270 tcacagccctgaacaaa 251 attatttccccaggccg 271 accttttaaaagagcat CCR7 probes 272 cggtaaaaccacacagga 296 caggtccatgacgctctc 273 cagcacgcttttcattgg 297 aatgacaaggagagccac 274 ttgacacaggcatacctg 298 tgtaatcgtccgtgacct 275 ccactgtggtgttgtctc 299 aagactcgaacaaagtgt 276 cgcacgtccttcttggag 300 ggaggaaccaggctttaa 277 acaaatgatggagtacat 301 attgcccagtaggcccac 278 ataggtcaacacgaccag 302 tggtcttgagcctcttga 279 ttgagcaggtaggtatcg 303 tgtaggcccagaagggaa 280 cgaagacccaggacttgg 304 tgagcttgcaaaagtgga 281 gctcatcttgtagatggc 305 taggagcatgccactgaa 282 gtcaatgctgatgcaaag 306 ctggacgatggccacgta 283 ggacagcttgctgatgag 307 gcactgtggctagtatcc 284 tacaggagctctgggatg 308 ctgctcctctggaggtca 285 agagcatcgcatcgcttg 309 ctccacatgctctgtgat 286 ccacctggatggtgataa 310 cagaaagccgatcaccat 287 gaagctcatggccagcag 311 gcggatgatgacaaggta 288 ctcaaagttgcgtgcctg 312 atcaccttgatggccttg 289 atgaagaccacgaccaca 313 attgtagggcagctggaa 290 cgtctgggccaggaccac 314 gctactggtgatgttgaa 291 ttgcttactgagctcaca 315 gacgtcgtaggcgatgtt 292 gacgcaggccaggctgta 316 gaaagggttgacgcagca 293 gacgccgatgaaggcgta 317 gaagagatcgttgcggaa 294 gcccaggtccttgaagag 318 gagctgctcctggctgag 295 gatgtgccgacaggaaga 319 ctccacactcatggagga cFos probes 320 tctgcaaagcagacttctca 344 tctgcaaagcagacttctca 321 ttcagcaggttggcaatctc 345 actctagtttttccttctcc 322 tcggtgagctgccaggatga 346 aggtcatcagggatcttgca 323 agacatctcttctgggaagc 347 agtcagatcaagggaagcca 324 tgaaggcctcctcagactcc 348 agggtcattgaggagaggca 325 acaggttccactgagggctt 349 tccatgctgctgatgctctt 326 atcaaagggctcggtcttca 350 tgatgctgggaacaggaagt 327 tcagagccactgggcctgga 351 tcagagccactgggcctgga 328 tgctgcatagaaggacccag 352 actgtgcagaggctcccagt 329 tctgtggccatgggccccat 353 tacaggtgaccaccggagtg 330 tgtaagcagtgcagctggga 354 taggtgaagacgaaggaaga 331 acagctggggaaggagtcag 355 tcattgctgctgctgccctt 332 agctgagcgagtcagaggaa 356 agtggcacttgtgggtgccg 333 tgtaatgcaccagctcgggc 357 gaagatgtgtttctcctctc 334 gtctacaggaaccctctagg 358 acagataaggtcctccctag 335 acagcctggtgtgtttcacg 359 ctttcaagtccttgaggccc 336 cttgagtccacacatggatg 360 atctccggaagaggtaagga 337 cactccatgcgttttgctac 361 gtgtcactgggaacaataca 338 ctaactaccagctctctgaa 362 aggcctggctcaacatgcta 339 agagaaaagagacacagacc 363 tatgagaagactaaggaga 340 cccaatagattagttaatgc 364 ccaggttaattccaataatg 341 caatttgaaaatatccagca 365 gttaaaatcagctgcactag 342 ccaggaacacagtagttatt 366 ctaatcagaacacactattg 343 cttagtataatattggtcat 367 ccagaaaataaagtcgtatc GFP probes 368 tcgcccttgctcaccat 392 accccggtgaacagctc 369 tcgaccaggatgggcac 393 ttacgtcgccgtccagc 370 gctgaacttgtggccgt 394 tcgccctcgccggacac 371 cgtaggtggcatcgccc 395 cttcagggtcagcttgc 372 ccggtggtgcagatgaa 396 agggcacgggcagcttg 373 agggtggtcacgagggt 397 actgcacgccgtaggtc 374 tcggggtagcggctgaa 398 cgtgctgcttcatgtgg 375 ggcggacttgaagaagt 399 acgtagccttcgggcat 376 agatggtgcgctcctgg 400 gccgtcgtccttgaaga 377 gcgcgggtcttgtagtt 401 cctcgaacttcacctcg 378 gttcaccagggtgtcgc 402 cccttcagctcgatgcg 379 cctccttgaagtcgatg 403 ccccaggatgttgccgt 380 ttgtactccagcttgtg 404 cgttgtggctgttgtag 381 gtcggccatgatataga 405 atgccgttcttctgctt 382 tcttgaagttcaccttg 406 ctcgatgttgtggcgga 383 agctgcacgctgccgtc 407 tgctggtagtggtcggc 384 tcgccgatgggggtgtt 408 ttgtcgggcagcagcac 385 gggtgctcaggtagtgg 409 tttgctcagggcggact 386 cgcttctcgttggggtc 410 gcaggaccatgtgatcg 387 ggcggtcacgaactcca 411 atgccgagagtgatccc 388 tcttgtacagctcgtcc 412 gaagccatggctaagct 389 tcctgctcctccacctc 413 tgggcagcgtgccatca 390 ctcctgggcacaagaca 414 tgacggtccatcccgct 391 agaagcacaggctgcag 415 tacacattgatcctagc HIV Gag RNA probes 416 ttaatactgacgctctcgca 440 catttatctaactctccccc 417 ccctggtcttaaccgaattt 441 ctgcttgcccatactatatg 418 aactgcgaatcgttctagct 442 atgtttctaacaggccagga 419 ccagtatttgtctacagcct 443 tgtctgaagggatggttgta 420 gagggtggctactgtattat 444 ctatcctttgatgcacacaa 421 gcttccttggtgtcttttac 445 gctcttcctctatcttctct 422 gctgtgcctttttcttactt 446 tttttcctgtgtcagctgct 423 gtaattttggctgacctggt 447 cctggatgttctgcactata 424 atggcctgatgtaccatttg 448 ctgaaagccttctcttctac 425 gctccttctgataatgctga 449 ggtgtttaaatcttgtgggg 426 tttgcatggctgcttgatgt 450 cttcctcattgatggtctct 427 tgcaatctatcccattctgc 451 tcatctggcctggtgcaata 428 tatgtcacttccccttggtt 452 gaagggtactagtagttcct 429 gtcatccatcctatttgttc 453 ctactgggataggtggatta 430 cccaggattatccatctttt 454 gtagggctatacatccttac 431 gtccttgtcttatgtccaga 455 catagtctctaaagggttcc 432 agagttttatagaaccggtc 456 tttacctcctgtgaagcttg 433 caacaaggtttctgtcatcc 457 aatctgggttcgcattttgg 434 gctggtcccaatgcttttaa 458 ggcatgctgtcatcatttct 435 aaactcttgctttatggccg 459 tacttggctcattgcttcag 436 ctgcatcattatggtagctg 460 cccttctttgccacaattga 437 ggctctgcaatttttggcta 461 tccaacagccctttttccta 438 tttggtgtccttcctttcca 462 cctgtctctcagtacaatct 439 gggccagattttccctaaaa 463 aagaaaattccctggccttc

The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated herein in their entireties. 

1. A method for detecting copies of at least one RNA molecule expressed in individual cells, comprising (a) providing a sample containing a population of cells that express specific receptors capable of initiating signal cascades leading to gene expression upon binding to them of ligands or stimulating molecules; (b) inducing gene expression in the cells ex vivo, either immediately or after culturing, by incubating the cells with at least one compound that will bind specific receptors and initiate gene expression; (c) fixing and permeabilizing the cells; (d) labeling copies of at least one RNA molecule expressed by the cells with a set of fluorescently labeled oligonucleotide hybridization probes, and washing away unbound probes; and (e) detecting cells having expression events by flow cytometry (FC), an expression event being one or more fluorescence measurements categorized by fluorescence intensity gating.
 2. The method of claim 1 wherein the step of inducing comprises stimulating T-cell receptor (TCR) signaling and down-stream gene expression by antigen presenting cells (APCs) that are present in the cell population or by an artificial APC (aAPC).
 3. The method of claim 2 wherein stimulation by APC or aAPC induction is for a period selected from the group consisting of from 30 minutes to 6 hours, from 6 to 24 hours, and from 24 to 72 hours.
 4. The method of claim 2 wherein the step of inducing includes co-stimulating with at least one monoclonal antibody (mAb).
 5. The method of claim 2, wherein the step of inducing includes co-stimulating with at least one monoclonal antibody (mAb) that is bound to the aAPC.
 6. The method of claim 1 wherein the compound is derived from a microorganism.
 7. The method of claim 1 wherein the compound comprises a peptide or mixture of peptides.
 8. The method of claim 2 wherein a peptide or mixture of peptides that associate with MHC class I or MHC class II molecules is added to the APC or aAPC.
 9. The method of claim 8, wherein the peptide or peptide mixture is present at a concentration between 1-20 μg/ml.
 10. The method of claim 1 wherein the at least one RNA molecule is encoded by a cytokine gene.
 11. The method of claim 10 wherein the gene encodes IL-2, TNFα, or IFNγ.
 12. The method of claim 1 wherein the stimulatory molecules are M. tuberculosis-derived immunogenic peptides.
 13. The method of claim 1 wherein the probe set comprises 20-60 probes, each singly labeled with the same fluorescent moiety.
 14. The method of claim 1 wherein the cell population is T cells obtained from human PBMCs, the at least one RNA molecule is encoded by a cytokine gene, cells in the cell population are induced by aAPC with one or more peptide-loaded MHC molecules that interact with TCR, and the probe set comprises 20-60 probes, each singly labeled with the same fluorescent moiety.
 15. The method of claim 1, wherein at least one detected cell having expression events is separated from cells not having expression events by fluorescence-activated cell sorting. 16-18. (canceled)
 19. The method of claim 15, wherein gene expression is measured in the separated cells.
 20. The method claim 19, wherein measurement of gene expression is accomplished by RT-PCR or a transcriptomic analysis of RNA in the cells.
 21. The method of claim 1 wherein the step of inducing comprises stimulating toll-like receptor signaling and down-stream gene expression with a microbial product or a synthetic compound, wherein the microbial product is a lipid, glycan, glycolipid, sulfolipid, glycoprotein, protein, peptide, or nucleic acid and wherein the stimulus is present at a concentration between 1-100 ng/ml, 100-1000 ng/m, or 1-20 μg/ml.
 22. (canceled)
 23. The method of claim 21, wherein stimulation is for a period selected from the group consisting of from 30 minutes to 6 hours, from 6 to 24 hours, and from 24 to 72 hours. 24-28. (canceled)
 29. The method of claim 1, wherein the compound is a cytokine.
 30. The method of claim 29, wherein the cytokine is one selected from the group consisting of IFNγ, IL-2, IL-15 and TNFα.
 31. (canceled)
 32. The method of claim 1 wherein more than one compound or stimulus is provided.
 33. The method of claim 32 wherein different stimuli are provided at different times.
 34. (canceled)
 35. A method for detecting copies of at least one RNA molecule expressed in individual cells, comprising (a) providing a sample containing a population of cells that express specific receptors capable of initiating signal cascades leading to gene expression upon binding to them of ligands or stimulating molecules; (b) fixing and permeabilizing the cells; (c) labeling copies of at least one RNA molecule expressed by the cells with a set of fluorescently labeled oligonucleotide hybridization probes, and washing away unbound probes; and (d) detecting cells having expression events by flow cytometry, an expression event being one or more fluorescence measurements categorized by fluorescence intensity gating.
 36. The method of claim 1, wherein the receptors are selected from the group consisting of toll-like receptors, NOD and NOD-Like receptors, G protein coupled receptors, polypeptide hormone receptors, cytokine receptors, B cell receptors, and T cell receptors.
 37. (canceled)
 38. The method of claim 1, wherein the population comprises cells selected from the group consisting of PBMCs, T-cells, lymphocytes, CD3⁺CD4⁺ T cells (T helper 1 or Th1 cells), CD3⁺CD4⁺ T cells (T helper 2 or Th2 cells), CD3⁺CD8⁺ T cells, Th17, Treg cells, NK cells, NKT cells, macrophages, dendritic cells, cells comprising endothelia and epithelia of various body organs, and cells of the nervous system. 